Expression of functional single-chain variable domain fragment antibody (scFv) against mycotoxin zearalenone in Pichia pastoris

Chang, Hyun-Joo; Choi, Sung‐Wook; Chun, Hyang Sook

doi:10.1007/s10529-008-9770-x

Cited by 18 publications

(9 citation statements)

References 14 publications

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“…Specific scFv have been developed for a number of mycotoxins including zearalenone (Chang, Choi, & Chun, 2008;Wang, Du, Lin, Huang, & Wang, 2008;Yuan et al, 1997), aflatoxin B 1 (Daly et al, 2002;Moghaddam, Løbersli, Gebhardt, Braunagel, & Marvik, 2001) and fumonisin B 1 (Lauer, Ottleben, Jacobsen, & Reinard, 2005;Min et al, 2010). There are also two reports of scFv specific for DON (Choi et al, 2004;Wang et al, 2007) and a report of a single-domain antibody fragment with an affinity to a related congener, 15-acetyl-deoxynivalenol (Doyle et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Production and characterization of a single chain variable fragment (scFv) against the mycotoxin deoxynivalenol

Maragos

Chen

2012

Food and Agricultural Immunology

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Deoxynivalenol (DON) is a mycotoxin produced by certain fungi that infest cereal grains. A hybridoma cell line producing a monoclonal antibody (Mab) was used as the starting point in the development of a recombinant single chain variable fragment antibody (scFv) recognising DON. The scFv and Mab were characterised using two immunoassay formats: competitive direct enzyme-linked immunosorbent assay (CD-ELISA) and biolayer interferometry (BLI). Using CD-ELISA the IC 50 s for DON were 36.1 and 13.8 ng/ml for assays based on the scFv and Mab, respectively. The cross-reactivity to DON analogs was very similar for the scFv and the Mab. The real-time binding of the antibodies to an immobilised DON-protein conjugate was also monitored. In competitive BLI assays the IC 50 s using the scFv and Mab were 68.3 and 15.8 ng/ml, respectively. The results suggest that sensitivity of assays, but not selectivity, was affected by removal of the constant regions of the Mab.

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Section: Introductionmentioning

confidence: 99%

Production and characterization of a single chain variable fragment (scFv) against the mycotoxin deoxynivalenol

Maragos

Chen

2012

Food and Agricultural Immunology

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“…There are different physical, chemical and biological strategies for Zearalenone (ZEA) decontamination, such as adsorbents [28] pulsed light technology [29], ozone gas oxidation [30], plantibodies [31] and cropping genetically modified maize that produce ZEA-degrading enzymes [32]. Biodegradation seems to be the most promising approach, with regards to ZEA decontamination.…”

Section: Discussionmentioning

confidence: 99%

A New Zearalenone Biodegradation Strategy Using Non-Pathogenic Rhodococcus pyridinivorans K408 Strain

et al. 2012

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Zearalenone (hereafter referred to as ZEA) is a nonsteroidal estrogenic mycotoxin produced by several Fusarium spp. on cereal grains. ZEA is one of the most hazardous natural endocrine disrupting chemicals (EDC) which induces hyper estrogenic responses in mammals. This can result in reproductive disorders in farm animals as well as in humans. Consequently, detoxification strategies for contaminated crops are crucial for food safety. In this study we have developed a bacterial based detoxification system using a non-pathogen Rhodococcus pyridinivorans K408 strain. Following 5 days treatment of ZEA with R. pyridinivorans K408 strain HPLC analyses showed an 87.21% ZEA-degradation efficiency of the bacterial enzyme systems. In another approach, the strain biotransformation ability has also been confirmed by a bioluminescent version of the yeast estrogen screening system (BLYES), which detected an 81.75% of biodegradability of ZEA, in a good agreement with the chemical analyses. Furthermore, the capacity of R. pyridinivorans to eliminate the estrogenic effects of ZEA was tested by using an immature uterotrophic assay. Prepubertal female rats were treated with vehicle (olive oil), 17β-estradiol, ZEA (0.1-1-5-10 mg/kg body weight) and LB broth containing 500 mg/l ZEA that has already been incubated with or without Rhodococcus pyridinivorans K408 strain. Uterine weights were measured and the mRNA level changes relating to apelin, aquaporin 5, complement component 2, and calbindin-3 genes were measured by qRT-PCR. These genes represent the major pathways that are affected by estromimetic compounds. Zearalenone feeding significantly increased the uterus weight in a dose dependent manner and at the same time upregulated complement component 2 and calbindin-3 expression as well as decreased apelin and aquaporin 5 mRNA levels comparable to that seen in 17β-estradiol exposed rats. In contrast, LB broth in which ZEA was incubated with Rhodococcus pyridinivorans K408 prior to the feeding did not display any estrogenic effect neither on uterine weight nor on the expression of estrogen-regulated genes. Consequently, the identification of Rhodococcus pyridinivorans K408 strain in ZEA biodegradation proved to be a very efficient biological tool that is able to eliminate the complete estrogenic effects of ZEA. It is also remarkable that this biotransformation pathway of ZEA did not result in any residual estrogenic effects.

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“…Many factors enhance the production level of recombinant protein in the P. pastoris expression system, such as the copy number of the cassette, site and mode of chromosomal integration of the expression cassette, AT content of cDNA, nature of the secretion signal, endogenous protease activity, host strain physiology, and media and growth conditions (Chang et al 2008). Culture conditions are critical parameters that affect cell growth and the yield of the recombinant product.…”

Section: Optimization Of Functional Expression Conditions Of Anti-bnpmentioning

confidence: 99%

Functional expression of recombinant anti-BNP scFv in methylotrophic yeast Pichia pastoris and application as a recognition molecule in electrochemical sensors

Maeng

Choi

Seung

et al. 2011

World J Microbiol Biotechnol

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Recent studies have revealed the potential of B-type natriuretic peptide (BNP) as a good prognostic marker for patients with heart failure. Antibodies against BNP are expected to be usefully employed in the diagnosis of heart failures. We established a more efficient method to produce functional anti-BNP, single chain variable fragment (scFv) using a eukaryotic expression system of Pichia pastoris. Although analysis of the N-terminal (NT) sequence of the expressed anti-BNP scFv indicated that the two Ste13 sites of the secreted anti-BNP scFv were not cleaved, the specificity of anti-BNP scFv was not affected significantly. The binding activity of anti-BNP scFv against other antigens, against four other antigens, NT probrain peptide (NT-pro BNP), atrial natriuretic peptide (ANP), carcinoembryonic antigen (CEA) and human serum albumin (HSA), was only one thousandth that of the original BNP antigen, which clearly demonstrated the specific binding of recombinant scFv toward BNP. The anti-BNP, scFv-based, electrochemical immunoassay exhibited excellent analytical performance with a detection limit of 1 fg/ml and a wide linear detection range from 1 to 10,000 fg/ml. The optimum culture conditions to obtain the maximum concentration of recombinant scFv were a pH range of 5.0-7.0 and an incubation temperature of 20°C. This anti-BNP scFv expressed in P. pastoris has the potential for promising applications in the diagnosis of heart failure.

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Expression of functional single-chain variable domain fragment antibody (scFv) against mycotoxin zearalenone in Pichia pastoris

Cited by 18 publications

References 14 publications

Production and characterization of a single chain variable fragment (scFv) against the mycotoxin deoxynivalenol

Production and characterization of a single chain variable fragment (scFv) against the mycotoxin deoxynivalenol

A New Zearalenone Biodegradation Strategy Using Non-Pathogenic Rhodococcus pyridinivorans K408 Strain

Functional expression of recombinant anti-BNP scFv in methylotrophic yeast Pichia pastoris and application as a recognition molecule in electrochemical sensors

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